Magnetic removal or identification of damaged or compromised cells or cellular structures

ABSTRACT

A method for magnetic cellular manipulation may include contacting a composition with a biological sample to form a mixture. The composition may include a plurality of particles. Each particle in the plurality of particles may include a magnetic substrate. The magnetic substrate may be characterized by a magnetic susceptibility greater than zero. The composition may also include a chargeable silicon-containing compound. The chargeable silicon-containing compound may coat at least a portion of the magnetic substrate. The biological sample may include cells and/or cellular structures. The method may also include applying a magnetic field to the mixture to manipulate the composition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/713,391 filed Sep. 22, 2017, which is a Continuation of U.S.application Ser. No. 13/974,139 filed Aug. 23, 2013, now U.S. Pat. No.9,804,153, issued Oct. 31, 2017, which claims priority to U.S.Provisional Applications 61/694,756 filed Aug. 29, 2012, the contents ofwhich are all incorporated by reference in their entireties.

BACKGROUND

Increasing the number or percentage of membrane intact, viable cells ina sample may improve overall sample quality, and may increase thesuccess of subsequent procedures. For example, an increase in the numberor percentage of membrane intact, viable sperm in a fresh orfrozen/thawed sample may improve overall sperm quality. Cleavage ratesfor both in vitro and in vivo fertilization procedures may be increased.Embryo quality may be enhanced and embryonic losses may be reduced,which may lead to increased pregnancy rates.

Magnetic cellular separation of apoptotic sperm has been achieved usingannexin V. However, it is desirable to remove sperm cells and spermcellular structures compromised in ways other than just apoptosis.Moreover, annexin V technology may be limited because the binding buffermay negatively affect sperm motility. Further, the cost of the reagentsmay potentially limit routine clinical application and adaptation of theprotocol to handle higher volumes and cell concentrations.

The present application appreciates that magnetic cellular manipulationmay be a challenging endeavor.

SUMMARY

In one embodiment, a composition for magnetic cellular manipulation isprovided. The composition may include a plurality of particles. Eachparticle in the plurality of particles may include a magnetic substrate.The magnetic substrate may be characterized by a magnetic susceptibilitygreater than zero. Each particle in the plurality of particles may alsoinclude a chargeable silicon-containing compound. The chargeablesilicon-containing compound may coat at least a portion of the magneticsubstrate.

In another embodiment, a method for magnetic cellular manipulation isprovided. The method may include contacting a composition with abiological sample to form a mixture. The composition may include aplurality of particles. Each particle in the plurality of particles mayinclude a magnetic substrate. The magnetic substrate may becharacterized by a magnetic susceptibility greater than zero. Eachparticle in the plurality of particles may include a chargeablesilicon-containing compound. The chargeable silicon-containing compoundmay coat at least a portion of the magnetic substrate. The biologicalsample may include cells and/or cellular structures. The method may alsoinclude applying a magnetic field to the mixture to manipulate thecomposition.

In one embodiment, a kit for magnetic cellular manipulation is provided.The kit may include instructions. The instructions may includecontacting a composition with a biological sample to form a mixture. Theinstructions may also include applying a magnetic field to the mixtureto manipulate the composition. The kit may also include the composition.The composition may include a plurality of particles. Each particle inthe plurality of particles may include a magnetic substrate. Themagnetic substrate may be characterized by a magnetic susceptibilitygreater than zero. Each particle in the plurality of particles mayinclude a chargeable silicon-containing compound. The chargeablesilicon-containing compound may coat at least a portion of the magneticsubstrate.

Additional objects, advantages, and novel features of the describedmethods and processes will be set forth, in part, in the descriptionthat follows; and, in part, will become apparent to those skilled in theart upon examination of the following; or may be learned by practice ofthe described methods and processes. The objects and advantages of thedescribed methods and processes may be realized and attained by means ofthe instrumentalities and combinations particularly pointed out, as wellas those items shown by inference.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures, which are incorporated in and form a part ofthe specification, illustrate one or more embodiments of the describedmethods and processes as related to sperm cells, and, together with thedescription, serve to explain the broad general cellular principles ofthe described methods and processes.

FIG. 1A shows flow cytometry analysis of an unlabeled ejaculate samplebefore example magnetic particle treatment, plotted as side lightscatter (SSC-H) as a function of forward light scatter (FSC-H)measurements;

FIG. 1B shows flow cytometry analysis of an unlabeled ejaculate samplebefore example magnetic particle treatment, plotted as fluorescenceheight (FL2-H) in counts, with nearly all cells live (M1);

FIG. 1C shows flow cytometry analysis of an unlabeled ejaculate samplebefore example magnetic particle treatment, plotted as a two-parameterdot plot of two fluorescence height measurements, FL2-H and FL1-H;

FIG. 1D shows flow cytometry analysis of an ejaculate sample beforeexample magnetic particle treatment, labeled with propidium iodide,plotted as side light scatter (SSC-H) as a function of forward lightscatter (FSC-H) measurements;

FIG. 1E shows flow cytometry analysis of an ejaculate sample beforeexample magnetic particle treatment, labeled with propidium iodide,plotted as fluorescence height (FL2-H) in counts, showing original deaduntreated cells (M2, 81.32%) and original live cells (Ml, 18.68%);

FIG. 1F shows flow cytometry analysis of an ejaculate sample beforeexample magnetic particle treatment, labeled with propidium iodide,plotted as a two-parameter dot plot of two fluorescence heightmeasurements, FL2-H and FL1-H;

FIG. 1G shows flow cytometry analysis of an ejaculate sample withparticles but before magnetic treatment at 34° C., plotted as side lightscatter (SSC-H) as a function of forward light scatter (FSC-H)measurements;

FIG. 1H shows flow cytometry analysis of an ejaculate sample withparticles but before magnetic treatment at 34° C., plotted asfluorescence height (FL2-H) in counts, showing dead particle treatedcells (M2, 14.73%) and live particle treated cells (Ml, 85.27%);

FIG. 1I shows flow cytometry analysis of an ejaculate sample withparticles but before magnetic treatment at 34° C., plotted as atwo-parameter dot plot of two fluorescence height measurements, FL2-Hand FL1-H;

FIG. 1J shows flow cytometry analysis of an ejaculate sample withparticles but before magnetic treatment at 25° C., plotted as side lightscatter (SSC-H) as a function of forward light scatter (FSC-H)measurements;

FIG. 1K shows flow cytometry analysis of an ejaculate sample withparticles but before magnetic treatment at 25° C., plotted asfluorescence height (FL2-H) in counts, showing dead particle treatedcells (M2, 18.32%) and live particle treated cells (M1, 81.68%);

FIG. 1L shows flow cytometry analysis of an ejaculate sample withparticles but before magnetic treatment at 25° C., plotted as atwo-parameter dot plot of two fluorescence height measurements, FL2-Hand FL1-H;

FIG. 2A shows zeta potential analysis details of example particlesdescribed herein as re-suspended in storage buffer at −28.2 mV;

FIG. 2B shows zeta potential analysis details of example particlesdescribed herein as re-suspended in storage buffer at −28.2 mV, as aplot of phase versus time in seconds;

FIG. 3A shows zeta potential analysis details of example particlesdescribed herein as re-suspended in storage buffer at −22.4 mV;

FIG. 3B shows zeta potential analysis details of example particlesdescribed herein as re-suspended in storage buffer at −22.4 mV, as aplot of phase versus time in seconds;

FIG. 4A shows zeta potential analysis details of example particlesdescribed herein as re-suspended in TALP buffer at −24.6 mV;

FIG. 4B shows zeta potential analysis details of example particlesdescribed herein as re-suspended in TALP buffer at −24.6 mV, as a plotof phase versus time in seconds;

FIG. 5A shows zeta potential analysis details of example particlesdescribed herein as re-suspended in MES buffer at −16.5 mV;

FIG. 5B shows zeta potential analysis details of example particlesdescribed herein as re-suspended in MES buffer at −16.5 mV, as a plot ofphase versus time in seconds;

FIG. 6A shows zeta potential analysis details of example particlesdescribed herein as re-suspended in dH2O −26.6 mV;

FIG. 6B shows zeta potential analysis details of example particlesdescribed herein as re-suspended in dH2O −26.6 mV, as a plot of phaseversus time in seconds;

FIG. 7A shows zeta potential analysis details of example particlesdescribed herein as re-suspended in TRIS buffer at −42.4 mV;

FIG. 7B shows zeta potential analysis details of example particlesdescribed herein as re-suspended in TRIS buffer at −42.4 mV, plotted asmobility in μmem/Vs;

FIG. 7C shows zeta potential analysis details of example particlesdescribed herein as re-suspended in TRIS buffer at −42.4 mV, plotted asfrequency shift in Hz;

FIG. 7D shows zeta potential analysis details of example particlesdescribed herein as re-suspended in TRIS buffer at −42.4 mV, as a plotof phase versus time in seconds;

FIG. 7E shows zeta potential analysis details of example particlesdescribed herein as re-suspended in TRIS buffer at −42.4 mV, plotted aszeta potential voltage and current versus time in seconds; and

FIG. 7F shows zeta potential analysis details of example particlesdescribed herein as re-suspended in TRIS buffer at −42.4 mV, plotted asa histogram.

DETAILED DESCRIPTION

The described methods and processes may include a variety of aspects,which may be combined in different ways and may be applied to differingcells or cellular materials. The following descriptions are provided tolist elements and describe some of the embodiments of the describedmethods and processes. These elements are listed with initialembodiments and are shown in examples of an embodiment relative to spermas but one initial example. However, it should be understood that eachand every permutation and combination of all aspects disclosed may beapplied in any manner and in any number to create additional embodimentsfor additional cells or cellular structures. The variously describedexamples and preferred embodiments should not be construed to limit thedescribed methods and processes to only the explicitly describedsystems, techniques, and applications. Further, this description shouldbe understood to support and encompass descriptions and claims of all ofthe various embodiments, systems, substances, elements, techniques,methods, devices, and applications with any number of the disclosedelements, with each element alone, and also with any and all variouspermutations and combinations of all elements in this or any subsequentapplication.

Briefly described, embodiments of the described methods and processesinclude a method for removing or identifying cells or cellularstructures having damaged membranes from those with intact membranes,thereby enriching sample or cellular viability. The method may beapplied to cells such as contained in freshly collected samples, afterdilution, during and after cooling, or during and after other cell orsystem procedures that may be employed prior to cryopreservation, or tofrozen/thawed cell samples. The method may also be used for samples thatmay be used immediately. The method may also be used for samples thatmay be held for a period of time or extended in buffers or othersubstances. For example, the method may also be used for samples thatmay be held at 4° C. to 40° C. for at least about 12, 24, 30, 36, 48,60, 72 hours or more. The method may also be used for samples thatinclude but are not limited to samples that have an osmolarity of250-375 mOsm. The enriched cell populations may be used for routineprocedures, prior to or after other processing techniques, prior to orafter shipment of samples, and prior to or after long-termcryopreservation or other processes.

Embodiments related to sperm may include a method for removing spermhaving damaged membranes from those with intact membranes to enrichsperm viability of a sperm sample. The method may be applied to spermcontained in freshly collected neat ejaculates, after dilution, duringand after cooling, or during and after other semen processing proceduresthat may be employed prior to cryopreservation, or to frozen/thawedsperm. The method may also be used for neat or extended sperm samples tobe used immediately. The method may also be used for neat or extendedsperm samples held up to 30 h at 4° C. to 40° C., or extended in spermrich buffers that have an osmolarity of 250-375 mOsm. The enriched spermpopulations may be used for routine artificial insemination, prior to orafter sperm sexing techniques, prior to or after shipment of semen forroutine or sperm sexing purposes or cryopreservation purposes, or for invitro fertilization, for all mammalian sperm.

In some embodiments, damage to the membranes of intact cells may bereduced by removing known harmful effects caused by damaged cells. Forexample, DNA fragmentation, oxidative damage caused by peroxidation, andthe premature release of proteolytic and hydrolytic enzymes may beexamples of effects attributable to membrane damage. Damage to cellsample integrity may reduce lifespan both in vitro and in vivo, mayreduce desired cellular functional ability, and may cause poor resultantcapabilities.

With respect to sperm as one, non-limiting example, damage to themembranes of intact sperm may be reduced by removing known harmfuleffects caused by damaged sperm. For example, DNA fragmentation,oxidative damage caused by peroxidation, and the premature release ofproteolytic and hydrolytic enzymes may be examples of sperm damagecaused by membrane damaged sperm. Damage to spermatozoal integrity mayreduce sperm lifespan both in vitro and in vivo, may reducefertilization ability, and likely causes poor embryo quality, which maybe a major source of infertility in mammals.

Mammalian sperm with good fertility may exhibit a high frequency ofmorphologically-normal, viable sperm. Current procedures for semenprocessing for sex selection, cooling, or cryopreservation may havedetrimental effects on the metabolism and motility of sperm, as well ason the status of sperm membrane domains. The net result of these effectsmay be reduced sperm functionality. Magnetic removal of damaged orcompromised cells or cellular structures may reduce a detrimental effecton the quality of live and normal sperm that may be caused by dead andabnormal sperm.

The pH of the medium suspending the cells may affect the charge ofproteins comprising the cells. Proteins may function as dipolar ions,for example, due to the ionization of the various R groups of the aminoacids that make up their primary structure. Medium pH may affectinteractions between such proteins. For example, capacitation of spermmay involve removal of seminal coating proteins absorbed on the sperm'ssurface membrane. Increasing the pH of the capacitating medium from maybe expected to alter the binding of proteins to the sperm's surface.Altering the binding of proteins to the sperm's surface may cause or beassociated with capacitation. For example, the capacitating medium mayhave a baseline pH between about 6.5 and 8.5, or in some examplesbetween about 7.2 and about 8.4. The capacitating medium may increase inpH in association with or in causation of successful capacitation. Theincrease in pH of the capacitating medium in association with or incausation of successful capacitation may be, in pH units, at least about0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9,or 1. In some examples, the increase in pH in association with or incausation of successful capacitation may be about 0.36 pH units.

Ionic components of the culture medium may influence mammalian spermmotility and a sperm's ability to penetrate an oocyte. The pH of themedium may cause the ionization of substances within the medium, forexample, including intrinsic sperm membrane proteins and extrinsic,absorbed seminal plasma proteins. The pH of the medium may determinemany aspects of the structure and function of biological macromolecules,including enzyme activity, and the behavior of cells.

The net charge on a cell surface may be affected by pH of thesurrounding environment and may become more positively or negativelycharged due to the loss or gain of protons. At or near physiologicalsperm pH, the net surface charge may be negative. Biological membranes,including sperm, may be negatively charged in physiological pH, mainlyas a result of the presence of acidic phospholipids. About 10-20% of thetotal membrane lipids may be anionic. Because the membrane may beexposed to surrounding aqueous buffer, specific interactions with outermedium components may occur. Charged groups of membrane components andsolutions ions may be involved in the resulting equilibria. Theequilibria may be affected by different factors and processes leading toa membrane surface charge density variation. The net charge may be alsoinfluenced by membrane composition, ionic strength of electrolytes, andsolution pH.

Viable mammalian sperm may have a net negative surface charge at theplasma membrane. As sperm undergo capacitation followed by the acrosomereaction, net charge may become less negative and more positive. The netcharge may become less negative and more positive due to, for example,loss of negative groups, such as sialic acid groups. Capacitation may becharacterized by the removal of coating materials from the spermsurface. Capacitation may be the penultimate step in fertilization,resulting in an increased permeability of the plasma membrane to Ca²⁺ions, and allowing the sperm to undergo the acrosome reaction or deathif fertilization does not occur. A viable mature human sperm may have anegative zeta potential of −16 to −20 mV (differential potential betweenthe sperm membrane and its surroundings), which may decrease withcapacitation to become more positively charged or near zero.

Sperm pre-capacitation may result in ova fertilization failure. Further,damage to sperm chromatin may result in poor embryo quality. Becausefertilization, as one example of many other cellular functions in thisclass, may be a time-sensitive event and good embryo quality may beessential for timely embryo development, both may be adversely affectedby sperm quality. Factors released from damaged sperm or other cells maybe partly responsible for further cellular damage to the remainingsubpopulation of cells such as normal sperm. For example, freshly killeddead sperm may have reduced livability of sperm in diluted bovine semen.Further, freshly ejaculated sperm subjected to elevated temperaturesbefore ejaculation may exhibit high reactive oxidative species levels.Thus, the toxic effect of dead cells, including but not limited tosperm, may be due to amino acid oxidase activity. Dead and abnormalcells such as sperm may have toxic effects on companion cells. Dead andabnormal cells may reduce cellular sample functionality such asfertility.

In various embodiments of the described methods and processes, carboxylgroup functionalized, silane-coated magnetic particles may be usedranging in physical diameter from 300 nm to 800 nm and an averagehydrodynamic diameter of about 330 nm. In some embodiments, the carboxylgroup functional silane coated magnetic particles may be used withoutfurther surface manipulation since the carboxyl group on the silanecontributes to the particles having a net negative electrical charge orzeta potential. Sperm may increase in intracellular pH withcapacitation, which may cause the membrane of the capacitated and deadsperm to lose net negative zeta potential and shift toward a neutral(zero) or more positive zeta potential. The negatively charged magneticparticles may bind specifically to compromised, damaged, or dead sperm.In addition, the silane surface may also be conjugated to othersubstances such as by standard EDC/NHS chemistry. In some examples,EDC-mediated coupling efficiency may be increased by adding the presenceof substances such as amine reactive esters for the conversion ofcarboxyl groups to amines.

In various embodiments, the described processes and methods may be usedto differentiate necrotic, apoptotic, and normal cells. In the examplediscussed, the carboxyl modified silane surface binds to the membrane ofthe dead and dying sperm through an electrical charge interaction knownas zeta potential. Material may spontaneously acquire a positive ornegative surface electrical charge when brought into contact with apolar medium (e.g., water). For example, an interface in deionized watermay be negatively charged. An ionization of surface groups to form asurface electrical charge may be observed with metal oxide surfaces(M-OH) as well as materials that may contain carboxyl and/or aminogroups, such as proteins, ionic polymers, and polyelectrolytes.Ionization and/or dissociation, degree of charge development, netmolecular charge, and sign, either positive or negative, may depend onthe pH of the surrounding medium.

The conjugation of carboxyl group functional magnetic particles mayadditionally be applied to, for example, fluorescent stains. SYBR-14 andbis-benzimide are example membrane permeable stains that may be used todistinguish cells from other background substances. In the sperm cellembodiment, such fluorescent stains may distinguish sperm cells fromdiluent particles frequently found in extenders employed in non-frozenstorage or cryopreservation of sperm. Other examples of fluorescentprobes may include JC-1 and rhodamine 123, which may be used to assessthe respiration rate of cell mitochondria; or fluorescently labeledagglutins from the pea (PSA) or peanut (PNA) that may be used to detectacrosome-reacted cells such as sperm. Other labels include but are notlimited to acridine orange (e.g., to remove apoptotic cells);7-aminoactinomycin D (7-AAD), which may be also a DNA intercalatingagent in double stranded DNA with a high affinity for GC rich regions;food coloring such as Allura Red (FD&C Red #40), Sunset Yellow (FD&CYellow #6), Indigo carmine (FD&C Blue #2), and Fast Green FCF (FD&C #3).

The cell plasma membrane may cover the entire cell and may have distinctmembrane compartments, such as, in the sperm cell example, the head,middle and principal portions. Since the plasma membrane may be composedof distinct membrane compartments, different stains may be used alone orin combination with other stains to assess the integrity of thedifferent plasma membrane compartments.

In some embodiments of the described methods and processes, cells withvarying degrees of membrane damage may be labeled with magneticparticles containing a charged surface. This may be in contrast to theuse of annexin-V/microbead magnetic cell sorting procedures that fail toidentify and/or remove pre-capacitated or acrosomal reacted spermbecause the PS does not become externalized in these examples. Whenmembrane damaged sperm or cellular structures labeled with the surfacecharged magnetic particles are placed in a magnetic field, such cells orcellular structures may be eliminated from the general population. Theresultant harvested sub-population of viable cells, perhaps such assperm, may be further processed for cryopreservation, non-frozentransport and storage, functional utilization (such as sex selection forsperm), or used in related aspects, perhaps such as assistedreproductive technologies (ARTs) for sperm or the like.

Embodiments of the described methods and processes may be used with anytype of magnetically identifying separating apparatus, including but notlimited to devices incorporating columns, such as magnetic-activatedcell sorting (MACS) products, devices using simple magnetic fieldsapplied to test tubes or containers, or high throughput magneticdevices.

Targeted dead and dying cells labeled with magnetic particles andsubjected to magnetic cell separation in an open, column-less magneticsystem may be removed more efficiently and in greater numbers per timeunit compared to flow cytometry. Magnetic cell separation may beutilized with no internal operating pressure, or if pressurized, a lowerinternal operating pressure; and the stream of fluid containing thecells may avoid being broken into cell damaging droplets as for flowcytometry. Further, the sheath fluid for flow cytometry may be generallya salt-based, lipoprotein-deficient physiological medium. Magnetic cellseparation may allow some cells, such as sperm, to be bathed innutrient-rich buffers that may promote and prolong cell viability duringthe separation procedure.

The described methods and processes may remove necrotic cells orcellular structures that have been traumatized during cell processingprocedures such as cryopreservation, centrifugation, and staining.Necrotic damage may occur by different cellular processes than thatcaused in one example by sperm senescence, which may be a naturallyoccurring cause of cellular death.

In other magnetic cell separation applications, embodiments of thedescribed methods and processes may be used to label cells uniquely,such as sperm, with one or more fluorochrome stains, targeting aspecific cell or sperm attribute. The targeted cell or sperm cell may beselectively killed or rendered non-functional, with an energy source,including but not limited to an electrical charge or pulse of laserlight. The described methods and processes may be used to magneticallylabel and ultimately remove the non-functional cell or sperm throughmagnetic cell separation procedures. The resultant desiredsub-population of harvested cells may be selected for membraneintactness (viability) as well as for specific cellular attributes,including but not limited to, in the sperm example, chromosomal sexselection.

In various embodiments, a composition for magnetic cellular manipulationis provided. The composition may include a plurality of particles. Eachparticle in the plurality of particles may include a magnetic substrate.The magnetic substrate may be characterized by a magnetic susceptibilitygreater than zero. Each particle in the plurality of particles may alsoinclude a chargeable silicon-containing compound. The chargeablesilicon-containing compound may coat at least a portion of the magneticsubstrate.

“Magnetic susceptibility” means the response of a sample, such as themagnetic substrate, to an externally applied magnetic field. Forexample, a magnetic susceptibility of less than or equal to zero may beassociated with diamagnetism. A magnetic susceptibility of greater thanzero may be associated with magnetic properties other than diamagnetism.For example, in various embodiments, the magnetic substrate may becharacterized by one or more of paramagnetism, superparamagnetism,ferromagnetism, or ferrimagnetism. The magnetic substrate may include ametal oxide, such as a transition metal oxide, for example, an ironoxide. In some examples, the magnetic substrate may include Fe₃O₄.

A “chargeable silicon-containing compound” is any silicon containingmolecule, polymer, or material that may be caused to acquire or hold acharge, e.g., via functionalization with charged or chargeable moieties.Chargeable/charged moieties may include, but are not limited to, species(and ions thereof) of: metals; oxides; carboxylates; amines; amides;carbamides; sulfates; sulfonates; sulfites; phosphonates; phosphates;halides; hydroxides; and combinations thereof. For example, thechargeable silicon-containing compound may include2-(carbomethoxy)ethyltrimethoxysilane.

In various examples, the composition may include a zeta potentialcharge. For example, the chargeable silicon-containing compound mayinclude a negative zeta potential charge. The chargeablesilicon-containing compound may include a positive zeta potentialcharge. In some examples, at least a portion of the plurality ofparticles may include a first zeta potential charge. The portion of theplurality of particles may form a complex with one or more cells orcellular structures that include a second zeta potential charge. Thesecond zeta charge may be opposite in sign compared to the first zetacharge. In several embodiments, at least a portion of the plurality ofparticles may include a negative zeta potential charge. The portion ofthe plurality of particles may form a complex with one or more spermcells or sperm cellular structures that include a positive zetapotential charge.

In various embodiments, the plurality of particles may include at leastone of a protein, an antibody, and a dye. The protein, antibody, or dyemay be conjugated to the chargeable silicon-containing compound. Forexample, the chargeable silicon-containing compound may include2-(carbomethoxy)ethyltrimethoxysilane, and the protein, antibody, or dyemay be conjugated to the 2-(carbomethoxy) group, e.g., via an amidebond.

In another embodiment, a method for magnetic cellular manipulation isprovided. The method may include contacting a composition with abiological sample to form a mixture. The composition may include aplurality of particles. Each particle in the plurality of particles mayinclude a magnetic substrate. The magnetic substrate may becharacterized by a magnetic susceptibility greater than zero. Eachparticle in the plurality of particles may include a chargeablesilicon-containing compound. The chargeable silicon-containing compoundmay coat at least a portion of the magnetic substrate. The biologicalsample may include cells and/or cellular structures. The method may alsoinclude applying a magnetic field to the mixture to manipulate thecomposition.

A “biological sample” may include any natural or prepared compositionthat includes the cells and/or cellular structures. Natural samples mayinclude, for example, biological fluids containing cells or cellularstructures, such as blood, lymphatic fluids, intestinal fluids,intercellular fluids, sweat, tears, urine, semen, mucosal secretions,synovial fluid, and the like. Natural samples may include fluidstypically free of cells or cellular structures, but which may includecells or cellular structures as part of injury, illness, genetic defect,or other pathological conditions. Prepared samples may include anybiopsy, tissue homogenate, or other prepared form of biological tissue.Typically, the biological sample will include at least one cell orcellular structure characterized by a zeta potential charge. Thebiological sample may include at least two or more cells or cellularstructures characterized by zeta potential charges differing in sign orcharge density. For example, a biological sample may include a firstcell characterized by a first zeta potential charge and a second cellcharacterized by a second zeta potential charge opposite in sign to thefirst zeta potential charge.

In some embodiments, the method may include causing the chargeablesilicon-containing compound to acquire a first zeta potential charge.The first zeta potential charge may be opposite in sign compared to asecond zeta potential charge comprised by the cells and/or cellularstructures in the biological sample. Causing the chargeablesilicon-containing compound to acquire the first zeta potential chargemay include contacting the chargeable silicon-containing compound to apolar medium, as described herein.

In several embodiments, the method may include causing the compositionand at least a portion of the cells and/or cellular structures in thebiological sample to form a complex. Applying the magnetic field to themixture to manipulate the composition may manipulate the complex.

In some embodiments, at least a portion of the plurality of particlesfurther comprises at least one of a protein, an antibody, and a dye.

In several embodiments, the biological sample may include viable cellsand damaged or compromised cells or cellular structures. The compositionmay selectively form a complex with one of the viable cells or thedamaged or compromised cells or cellular structures, for exampleaccording to a first zeta potential charge on the composition and anopposite second zeta potential charge on one of the viable cells or thedamaged or compromised cells or cellular structures. The method mayfurther include separating the viable cells from the damaged orcompromised cells or cellular structures by applying the magnetic fieldto the mixture. Because the composition may selectively form a complexwith one of the viable cells or the damaged or compromised cells orcellular structures, the portion of the biological sample forming thecomplex with the composition may be magnetically manipulated andseparated from portions of the biological sample not forming the complexwith the composition. The method may therefore be a method forselectively and magnetically separating portions of the biologicalsample according to zeta potential charge.

In various embodiments, the biological sample may include viable spermcells and damaged or compromised sperm cells or sperm cellularstructures. The composition may form a complex with the damaged orcompromised sperm cells or sperm cellular structures. The method mayfurther include separating the viable sperm cells from the complexincluding the damaged or compromised sperm cells or sperm cellularstructures by applying the magnetic field to the mixture. Because thecomposition may selectively form a complex with the damaged orcompromised sperm cells or sperm cellular structures, the complex withthe damaged or compromised sperm cells or sperm cellular structures maybe magnetically manipulated and separated from the viable sperm cells.The method may therefore be a method for selectively and magneticallyseparating viable sperm cells from the damaged or compromised spermcells or sperm cellular structures according to zeta potential charge.

In several embodiments, the method may include subjecting the spermsample to detection, for example fluorescence detection as describedherein.

In various embodiments, a kit for magnetic cellular manipulation isprovided. The kit may include instructions. The instructions may includecontacting a composition with a biological sample to form a mixture. Theinstructions may also include applying a magnetic field to the mixtureto manipulate the composition. The kit may also include the composition.The composition may include a plurality of particles. Each particle inthe plurality of particles may include a magnetic substrate. Themagnetic substrate may be characterized by a magnetic susceptibilitygreater than zero. Each particle in the plurality of particles mayinclude a chargeable silicon-containing compound. The chargeablesilicon-containing compound may coat at least a portion of the magneticsubstrate.

In some embodiments of the kit, the biological sample may include viablecells and damaged or compromised cells or cellular structures. Thecomposition may form a complex with one of the viable cells or thedamaged or compromised cells or cellular structures. The instructionsmay further include separating the viable cells from the damaged orcompromised cells or cellular structures by applying the magnetic fieldto the mixture.

In several embodiments of the kit, the composition may be configured forforming a complex with damaged or compromised sperm cells or spermcellular structures. The instructions may further include selecting thebiological sample comprising viable sperm cells and damaged orcompromised sperm cells or sperm cellular structures. The instructionsmay also include separating the viable sperm cells from the complexincluding the damaged or compromised sperm cells or sperm cellularstructures by applying the magnetic field to the mixture.

Having generally described the present method, more details thereof maybe presented in the following EXAMPLES. Although the examples involvesperm cells as the cell item, the selection or application is notintended to limit the scope of the described methods and processes, asother types of cells are valuable in applications of the generalteaching of the described methods and processes.

Example 1: Particle Preparation for Magnetic Staining of Dead/DamagedCells with Sperm as a Representative, Non-Limiting Example

Magnetic cores: Magnetic cores may be fabricated such as byco-precipitation of Fe₃O₄ with Fe₂O₃ so that the magnetic susceptibilityof the particles in a chosen magnetic field may be sufficiently high toprovide rapid separation of magnetically labeled cells from non-labeledcells. The core may be comprised of any magnetic material; possiblenon-limiting examples include: (1) ferrites such as magnetite, zincferrite, or manganese ferrite; (2) metals such as iron, nickel, orcobalt; and (3) chromium dioxide. In one embodiment, the iron cores arecomprised of magnetite (Fe₃O₄). In other embodiments the core may beextended to include substances such as other iron oxide basednanoparticle materials including composites having the general structureMFe₂O₄ (where M may be Co, Ni, Cu, Zn, Mn, Cr, Ti, Ba, Mg, or Pt).

Thus, in this one non-limiting example, a reaction chamber containing400 mL of dH₂O in a water kettle was warmed to 60° C. To the 400 mL ofwarmed dH₂O, 23.4 g of FeCl₃.6H₂O and 8.6 g of FeCl₂ or the like wasadded and the mixture was stirred under N₂ gas. To this solution, 30 mLof 25% NH₃.H₂O was added and mixing was continued under N₂ gas. Almostimmediately, the orange salt mixture turned to a dark brown/blacksolution. The heat was turned off and the ferrofluid slurry was allowedto cool while being vigorously stirred for 30 min. The precipitate wascollected magnetically and the supernatant was decanted. To themagnetically collected ferrofluid, 800 mL of dH₂O was added, swirled,and the magnetic collected process was repeated. The washing process wasrepeated four times to remove substantially all residual NH₃.H₂O and anynonmagnetic particles. The final wash step may include a solution of 800mL 0.02 M NaCl in dH₂O or the like. The collected iron core sizes werebetween approximately 3 and approximately 10 nm.

Coating of Iron Cores with a functionalizable surface: The final outerlayer may comprise a polymer coat that interacts with the aqueousenvironment and serves as an attachment site for proteins and ligands.Suitable polymers may include polysaccharides, alkylsilanes,biodegradable polymers such as, for example, poly(lactic acids) (PLA),polycaprolactone (PCL), and polyhydroxybutyrate-valerate (PHBV);composites, and polyolefins such as polyethylene in its differentvariants. More specifically, polysaccharide chains may include dextrans,arabinogalactan, pullulan, cellulose, cellobios, inulin, chitosan,alginates, and hyaluronic acid. Silicon containing compounds such asalkylsilanes may also be employed to encapsulate the magnetic core.Alkylsilanes suitable for embodiments of the described methods andprocesses, may include but are not limited to, n-octyltriethoxysilane,tetradecyltrimethoxysilane, hexadecyltriethoxysilane,hexadecyltrimethoxysilane, hexadecyltriacetoxysilane,methylhexadecyldiacetoxysilane, methyl-hexadecyldimethoxysilane,octadecyltrimethoxysilane, octadecyltrichlorosilane,octadecyltriethoxysilane, and 1,12-bis(trimethoxysilyl)dodecane. In oneexample, the ratio of iron to silicon containing compound coating may beapproximately 0.2. In other embodiments, the ratio of iron to siliconcontaining compound coating may be greater than about 0.2, such as about0.4 or 0.8, with a view toward completely coating the iron cores suchthat the iron cores may be removed from the cell suspension within themagnetic field. Undercoated particles may result in the free metal oxidecrystals which may be detrimental to cell viability. In still otherembodiments, the ratio of iron to silicon containing compound coatingmay be less than 0.2. Indeed, the iron concentration divided by thesilicon containing compound concentration may be from about 0.1 to about1.

For the examples of magnetic removal of dead/dying or compromised cellssuch as sperm, a silicon containing compound may be used to encapsulatethe iron cores.

The iron core precipitate may be allowed to settle. With theunderstanding that throughout this disclosure all amounts, times, andvalues may be varied up or down such as by 10%, 20%, 30%, or even 40% inany permutation or combination for some embodiments, 67.1 mg of theferrofluid were added to 100 mL of 10%2-(carbomethoxy)ethyltrimethoxysilane.2-(carbomethoxy)ethyltrimethoxysilane is yet another example of asilicon containing compound that is suitable for use in the describedmethods and processes as a silicon containing compound coating. The pHwas adjusted to 4.5 using >99.5% glacial acetic acid, and the suspensionwas reacted at 70° C. for 2 h under N₂ gas with vigorous mixing. Aftercooling, the particles may be magnetically collected and washed withdH₂O. After washing, the silane-coated magnetic nanoparticles may besuspended in 5 mL of 0.05 M 2-(N-morpholino)ethanesulfonic acid (MES)Buffer, TRIS Buffer, TALP buffer, or it may remain in the dH₂O until usefor separation. The resuspension buffer may be at a pH that retains orcreates a net negative zeta potential of the particles.

Iron concentration may be determined using Inductively CouplePlasma-Optical Electron Spectroscopy (ICP-OES), and the ironconcentration may be adjusted according to milligrams per milliliterneeded for optimal dead cell removal. The particles may have an averagehydrodynamic diameter of 300 nm, and need to be in a range of 300 to1000 nm to stay suspended in solution so that maximum interactionbetween the cells and particles is achieved by keeping the particles insuspension and not settling out due to larger sizes.

Coupling of proteins and ligands to the particle surfaces: In the eventthat the surface of the particles needs to be treated and conjugated toa protein or antibody, the following methods may be used. Periodatetreatment of dextran and other polymers are one method for theattachment of proteins due largely to the large number of reactivegroups that are available for modification. Mild sodium periodatetreatment may create reactive aldehyde groups by oxidation of adjacenthydroxyl groups or diols. Proteins, antibodies, streptavidin, andamino-modified nucleic acids may be added at high pH to allow amines toform Schiff bases with the aldehydes. The linkages may be subsequentlyreduced to stable secondary amine linkages by treatment with sodiumborohydride or sodium cyanoborohydride, which may reduce unreactedaldehyde groups to alcohols. Another method of coupling proteins to themagnetic nanoparticles may be to create stable hydrazine linkages. Forexample, a protein may be coupled to dextran using succinimidyl4-hydrazinonicotinate acetone hydrazone (SANH; Solulink Inc, San Diego,Calif.). The reaction may use five-fold less protein, and the resultingprotein density may appear as high as with other methods. The SANHreagent may allow more efficient and gentle coupling of ligands to thedextran surface. Ligand attachment on silica-coated magneticnanoparticles may be completed using (3-aminopropyl)triethoxysilane(APTS) to introduce amines on the particle surface while(3-mercaptopropyl)triethoxysilane (MPTMS) introduces SH groups. Theheterobifunctional coupling agent (Succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate) may then be used to linkthiols to the amines. As examples, amines on the particle surface may belinked to thiols on streptavidin molecules, and thiols on the particlesurface may be linked to amines on streptavidin. There are severalmethods of crosslinking proteins through chemical modifications known inthe art that may be used for the present embodiments of the describedmethods and processes. For this example, the carboxylic acidfunctionalized silane may attach proteins and ligands through EDCchemistry.

EDC Activation of COOH groups on Particle Surface Activation: Thesilanized particles were re-suspended in 0.05 M MES buffer, collectedmagnetically, and the supernatant may be aspirated and discarded.Another 5 mL of MES buffer (0.05 M, pH 4.7-5.2) per 10 mg of iron wasadded to the particles and the suspension was vigorously shaken.Particles were magnetically collected, and the supernatant was aspiratedand discarded. This step may be repeated two or so additional times.Frozen EDC was allowed to thaw at room temperature for 30 min. EDC (alsoknown as EDAC or EDCI, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide),commonly obtained as a hydrochloride, is a water soluble carbodiimide,which is typically employed at pH in the range between 4.0 and 6.0. EDCmay be used as a chemical crosslinker for collagen, reacting with thecarboxylic acid groups of the collagen polymer which may then bond tothe amino group in the reaction mixture.

1.6 mg of EDC/mg iron was added to the particle suspension and thesuspension was shaken vigorously. Each tube or the like containingparticles and EDC was be placed on a laboratory rocker at roomtemperature for 30 min. After 30 min., particles were magneticallycollected, and the supernatant was aspirated and discarded. Buffers ofvarying salt concentrations, molarities, including but not limited to,0.1 M to 1 M, and pH ranges from 10 to 4.7 may be used for proteinconjugation to the various surfaces. The function of each antibody,protein and ligand optimizes at different pH ranges and molarities, asis known in the art (Hermanson, Bioconjugate Techniques, 2008). In thisexample, the particle pellet was added to 0.05M MES buffer, particlesmay be magnetically collected, and supernatant may be aspirated anddiscarded. This step may be repeated three or so times. 10 mg ofprotein, ligand or stain was suspended in 0.05M MES buffer and added tothe particles so that the total labeling volume was about 5 mL per 10 mgof iron.

A stoichiometric balance of 1 mg of protein, ligand, or stain per 1 mgof iron was used for the coupling reaction. Some experiments suggestedthat the best binding of dead or compromised cells is concentrationdependent and may occur at about this concentration (with the abovepercentage variations applicable, of course). The range in antibody orprotein used may include, but is not limited to, 0.125 mg to 5 mg ofantibody per mg of iron. Tubes were shaken and placed on a laboratoryrocker at room temperature for 24 h, and particles were magneticallycollected. The supernatant was aspirated and discarded. Each particlesuspension was suspended in 5 mL of MES buffer. To each tube, 5 mL ofquenching solution (1M glycine, pH 8.0) was added and the tubes wereshaken vigorously. Quenching solutions may include, but are not limitedto, 2-mercaptoethanol, ethanolamine, glycine, UV exposure, sizeexclusion and magnetic collection. Each tube was placed on a laboratoryrocker for 30 min. at room temperature. After 30 minutes, 5 mL of washbuffer was added to each tube and shaken to mix. The particles weremagnetically separated, and the supernatant was aspirated and discarded.This step may be repeated 4 or so times. After the conjugation processwas complete, particles were magnetically collected, washed, andfiltered to obtain a size distribution of 50 to 400 nm. After the washsteps, each particle suspension was suspended in a buffer for theparticular cells, such as for sperm cells or other such cells, with a pH(6.0-8.0) and osmolarity (250-350) suitable for optimal sperm or othercell viability such as but not limited to TRIS solution, Sodium Citratesolution, TEST solution, egg yolk-TRIS (pH 6.5-7.4), egg yolk-sodiumcitrate (pH 6.5-7.4), egg yolk-TEST (pH 6.5-7.6), milk extenders (pH6.5-7.4), commercially available extenders marketed by IMVInternational, Maple Grove, Minn., USA and MiniTube GmbH, Vernona, Wis.,USA and chemically defied media including but limited to TALP (pH6.0-8.0) Tyrodes (pH 6.0-8.0) and Hepes BGM-3 (pH 6.0-8.0) so that theresultant working iron concentration was about 4 mg/mL as confirmed byInductive Coupled Plasma-Optical Emission Spectroscopy (ICP-OES).

The particles may be advantageously on the order of about 300 nm so thatinteraction between the particles and that of the damaged or dead cellsis maximized in solution. If particles are too large, such interactionmay not occur due to the settling effect of the larger sized particlesin solution. If particles are smaller than approximately 30 nm, they mayeither not be sufficiently magnetic and higher magnetic susceptibilitycore materials within a chosen magnetic energy field will have to begenerated, or these small particles may contribute to nonspecificbinding; that is, they may bind to viable cells as well as to dead anddying cells. If nonspecific binding relating to particle size isproblematic, particle size may either be increased, or a blocking agentdependent upon the particular cells involved, such as nonfat dried milkor serum albumin for sperm, may be added to the labeling buffer solutionto minimize such nonspecific binding.

The surface charged particles used in Examples 2-4 are comprised ofFe₃O₄ coated with 2-(carbomethoxy)ethyltrimethoxysilane, without furtheractivation or functionalization. The ratio of iron to silicon containingcompound coating is approximately 0.2.

Example 2: Removal of Damaged Cells

Removal of Dead and Dying Sperm from a Thawed Cryopreserved SemenSample.

Six straws of semen were obtained from liquid nitrogen and thawed in awater batch for 2 min. Contents of all six straws were emptied into one50 mL falcon tube (about 240 million sperm). About 10 mL of bovinesheath fluid was added and the cells were spun for 7 min at 1800 RPMs.The supernatant was decanted. The cell pellet was re-suspended in 2 mLof bovine sheath fluid and the cells were divided equally into sevenappropriately labeled tubes. To each tube (not the unlabeled control orthe original dead control), 0.1 mg of surface charged particles wereadded to each tube requiring particles. Samples were either incubated ina 34° C. water bath for 20 min or at room temperature for 20 min, todetermine whether uptake of particles is facilitated at a highertemperature. After the incubation period had expired, the cellsuspensions containing the particles and magnetically labeled cells wereplaced in a magnetic field. Once a magnetic pellet was collected, thenonmagnetic supernatant was aspirated and placed into another tube.Propidium iodide was added to the nonmagnetic fraction to label the deadand dying population to compare it to the original percent dead anddying cells that were not treated with particles. Cells were incubatedwith 100 μl of propidium iodide in the dark for 20 min and analyzed by aflow cytometer. The original dead percent was approximately 81% and wasreduced to 15% using the magnetic particle treatment (FIG. 1A-IL).

Removal of Dead and Dying Sperm from a Fresh Bull Semen Sample.

One ejaculate was collected from each of three bulls. The ejaculatesperm concentration and volume were recorded and 640×10⁶ cells perejaculate were divided into four aliquots. Cells were gently centrifugedat 5000 RPMs for 6 min and the seminal plasma was aspirated from thepellet with a pipette. Each cell pellet was re-suspended in 4 mL ofpre-warmed TRIS medium, so that the concentration of cells was 160×106cells/mL. One mL of each re-suspended cell pellet was pipetted into fourdifferent 50 mL conical tubes: 1) control 34° C., 2) control RT, 3)particle treated 34° C., and 4) particle treated RT. To each treatedsample, 100 μl of a 1.8 mg stock solution of net negative chargedmagnetic particles re-suspended in 600 μl of storage buffer (pH 7.4PBS+0.1% BSA) was added and placed at the appropriate temperature for 20min. After the 20 min incubation period had expired, for those samplescontaining particles, they were placed in front of a magnet for 1 min.The nonmagnetic fraction was aspirated out of the tube and placed intoan eppendorf tube. From each sample after each magnetic separation wascomplete, aliquots were analyzed by flow cytometry for the dead percentprior to particle treatment as well as the dead percent after particletreatment. The average increase in viable sperm after the particletreatment was 18.7% (a change from an average of 74.68% viable to 93.38%viable after treatment). Optimal separation may occur once sperm thatare susceptible have begun the process of capacitation and membranealteration and have begun to die. This happens once the pH of the spermhas increased by at least 0.36 pH units (Vredenburgh-Wilberg, W. I. andParrish, J. J. “Intracellular pH of Bovine Sperm Increases DuringCapacitation,” Molecular Reproduction and Development 40:490-502, 1995).

Results:

Bull A Control 34° C. 77.90% Bull A Treated 34° C. 94.90% Bull A ControlRT 77.60% Bull A Treated RT 95.37% Bull B Control 34° C. 78.73% Bull BTreated 34° C. 98.70% Bull B Control RT 83.53% Bull B Treated RT 97.87%Bull C Control 34° C. 66.60% Bull C Treated 34° C. 88.43% Bull C ControlRT 63.87% Bull C Treated RT 85.03%

Example 3: Removal of Damaged Sperm from Stallion Ejaculates

One ejaculate from each of three stallions was collected. Ejaculatesperm concentration was determined using a densimeter. Motility wasdetermined objectively by a Sperm Vision CASA System (MiniTube, Verona,Wis., USA). The pH of the raw ejaculate was measured. Two aliquots of160×10⁶ total sperm were removed from each ejaculate from each stallion,representing control and treated samples. For control samples, spermwere immediately re-suspended in 1 mL of Modified Whitten's medium (pH7.0) (Funahashi et al., 1996; Biology of Reproduction, 54:1412-1419) andheld at room temperature. For particle treated sperm, 160×10⁶ totalsperm from each stallion were added to 1 mL of particles from a 3.6mg/mL stock solution that had been collected and removed from a particlestorage medium and suspended in 1 mL of Modified Whitten's medium in a50-mL Falcon tube. The sperm/particle admixture was allowed to incubateat room temperature for 20 min. Following incubation, the tube wasplaced onto a magnet for 3 min. The non-magnetic fraction was removed byaspiration and transferred to a clean test tube. Total and progressivemotility of the enriched sperm from each stallion was determined usingthe Sperm Vision CASA System and compared to the initial motilitydeterminations.

All samples were then diluted to 25×10⁶ progressively motile sperm/mLwith E-Z Mixing CST (Animal Reproduction Systems, Chino, Calif., USA)and allowed to cool to 5° C. for 24 h. Following the 24 h coolingperiod, samples were warmed to 37.5° C. for 10 min and assayed for totaland progressive motility using the Sperm Vision CASA System.

Results:

0-h 0-h 24-h Cooled 24-h Cooled Total Progressive Total ProgressiveMotility % Motility % Motility % Motility % Stallion A Ejaculate pH:6.91 Control 78 59 50 45 Treated 88 73 60 50 Stallion B Ejaculate pH:6.96 Control 61 48 11 6 Treated 65 51 26 22 Stallion C Ejaculate pH:7.40 Control 75 72 53 47 Treated 88 82 61 58 OVERALL RESULTS 0-h 0-h24-h Cooled 24-h Cooled Total Progressive Total Progressive N Motility %Motility % Motility % Motility % Control 3 71.3 59.7 38 32.7 Treated 380.3 68.7 49 43.3

The removal of damaged/dead sperm from a fresh ejaculate, followed byextending and cooling sperm for 24 h resulted in an increase in bothtotal and progressive motile sperm in each treated sample compared tocontrols. The overall improvement in motility scores from treatedsamples was observed at both the 0 h (prior to cooling) and post-cool 24h period.

Example 4: Removal of Damaged Sperm from Stallion Ejaculates Prior toCryopreservation

On day 1, a single ejaculate was collected from each of two stallionsand divided into two aliquots, one for the untreated control and theother for the particle treated sample. The neat semen was immediatelydiluted 1:1 with Modified Whitten's medium. The diluted samples werecentrifuged for 7 min to remove the seminal plasma. The supernatant wasimmediately aspirated and discarded. The sperm pellet was suspended andsperm concentration was obtained. The untreated control aliquots werere-suspended to 40×10⁶ cells/mL in a French egg yolk/milk extendercontaining 5% glycerol. Control sperm were cooled for 2 h at 5° C.,packaged in 0.5 mL straws, and frozen over liquid nitrogen vapor. To thetreated aliquots, 1:1 mL of particles (3.6 mg/mL) re-suspended inModified Whitten's medium was added to 160×10⁶ total sperm contained in500 μL of Modified Whitten's medium. Treated samples were allowed toincubate with the particles for 20 min at room temperature. Followingparticle exposure time to the sperm, the particles were magneticallycollected and the nonmagnetic fraction was aspirated and dispensed intoa separate tube. Treated samples for both stallions were extended to atotal of 4 mL with a French egg yolk/milk extender containing 5%glycerol. Treated sperm were allowed to cool for 2 h at 5° C., and werepackaged in 0.5 mL straws and frozen over liquid nitrogen.

On day 2, a single ejaculate was collected from each of two stallionsand each ejaculate was divided into two aliquots: untreated control andparticle treated. The neat semen was diluted 1:1 with Modified Whitten'smedium, and centrifuged for 9 min. The seminal plasma was aspirated anddiscarded. For both control and treated samples, 80×10⁶ total sperm weredeposited into 275 μL Whitten's medium. Control samples were extended to40×10⁶ sperm/mL in a total of 2 mL with EZ Freezin-LE (AnimalReproduction Systems) a prepackaged Lactose/EDTA freezing extendercontaining 5% glycerol. Treated samples were allowed to incubate withthe particles for 20 min at room temperature. After the incubationperiod had expired, magnetically labeled cells were collected on amagnet and the nonmagnetic cells were aspirated and placed in anothertube. Treated samples were re-suspended to 40×10⁶ sperm/mL in a total of2 mL with EZ Freezin-LE. Extended semen from each stallion/treatment waspackaged in 0.5 mL straws and placed on a freezing rack. The freezingrack was placed inside of a styrofoam box containing a known depth ofliquid nitrogen so that the straws were in the vapor phase of thenitrogen and the lid was loosely placed over the top of the box.

Results:

CONTROL (%) TREATED (%) Total Progressive Total Progressive StallionMotility Motility Live Dead Motility Motility Live Dead Sammy 5 2 22 7845 30 70 30 Gunsmoke 15 5 14 86 40 10 75 25 Tinman 62 22 56 44 67 26 7327 Scotti 62 34 50 50 58 33 54 46 MEAN 0-h TOTAL AND PROGRESSIVEMOTILITY (%) and Live/Dead (%) Total Progressive Motility Motility LiveDead Control 36 16 36 64 Treated 52 25 68 32 N = 4 Stallions

Removal of damaged/dead stallion sperm prior to cryopreservationincreased 0 h post-thaw total and progressive motility by 44% and 62%respectively, compared to control sperm. The percentage of viable spermimmediately after thawing was increased 32 percentage points or 89% whendead and/or damaged sperm were removed prior to cryopreservation.Removal of compromised sperm prior to cryopreservation increased overallsperm quality.

Zeta potential measurements of carboxyl group containing silane coatedmagnetic particles in buffers such as TRIS, TALP, dH₂O, and storagebuffer were measured by a zeta sizer and the resulting net zetapotential is shown in FIGS. 2A-7F. Particles with carboxyl silanecoating in this example measure as a net negative zeta potential in eachbuffer condition and may be expected to bind to sperm having undergoneor undergoing capacitation and losing the net negative charge seen inviable sperm.

Parameters for FIGS. 1A-1C:

Quadrant Statistics File: unstaned.001 Log Data Units: Linear ValuesSample ID: unstained Patient ID: Tube: Untitled Panel: UntitledAcquisition Tube List Acquisition 09-Dec-11 Gate: G1 Date: Gated Events1000 Total Events: 12962 X Parameter: FL1-H (Log) Y Parameter: FL2-H(Log) Quad 9, 14 location: % % X Geo Quad Events Gated Total X Mean MeanY Mean Y Mean UL 3 0.03 0.02 7.17 7.13 17.80 17.78 UR 1 0.01 13.95 13.9529.96 29.96 LL 9996 99.96 77.18 2.76 2.59 3.22 2.88 LR 0 0.00 0.00 ****** *** ***

Parameters for FIGS. 1D-1F:

Quadrant Statistics File: unstaned.001 Log Data Units: Linear ValuesSample ID: unstained Patient ID: Tube: Untitled Panel: UntitledAcquisition Tube List Acquisition 09-Dec-11 Gate: G1 Date: Gated Events1000 Total Events: 12962 X Parameter: FL1-H (Log) Y Parameter: FL2-H(Log) Quad 9, 14 location: % % X Geo Quad Events Gated Total X Mean MeanY Mean Y Mean UL 8132 81.32 69.70 2.73 2.59 143.93 131.70 UR 4 0.04 0.0515.06 13.04 128.51 120.79 LL 1863 16.63 15.97 3.02 2.83 3.57 3.35 LR 10.01 0.01 9.82 9.82 9.22 9.22

Parameters for FIGS. 1G-1I

Quadrant Statistics File: non-mag 34.006 Log Data Units: Linear ValuesSample ID: non-mag 34 Patient ID: Tube: Untitled Panel: UntitledAcquisition Tube List Acquisition 09-Dec-11 Gate: G1 Date: Gated Events1000 Total Events: 31123 X Parameter: FL1-H (Log) Y Parameter: FL2-H(Log) Quad 9, 14 location: % % X Geo Quad Events Gated Total X Mean MeanY Mean Y Mean UL 1473 14.73 4.70 2.98 2.74 49.18 43.42 UR 3 0.03 0.01319.23 82.54 34.20 29.60 LL 8516 85.16 27.36 2.93 2.64 3.87 3.38 LR 80.06 0.03 21.04 14.19 7.42 6.31

Parameters for FIGS. 1J-1L

Quadrant Statistics File: non-mag RT.005 Log Data Units: Linear ValuesSample ID: non-mag RT Patient ID: Tube: Untitled Panel: UntitledAcquisition Tube List Acquisition 09-Dec-11 Gate: G1 Date: Gated Events1000 Total Events: 31123 X Parameter: FL1-H (Log) Y Parameter: FL2-H(Log) Quad 9, 14 location: % % X Geo Quad Events Gated Total X Mean MeanY Mean Y Mean UL 1832 18.32 6.26 2.65 2.52 52.41 45.04 UR 0 0.00 0.00*** *** *** *** LL 8168 81.68 27.92 2.70 2.42 3.63 3.12 LR 0 0.00 0.00*** *** *** ***

Parameters for FIGS. 2A and 2B

Measurement Details Sample Name: Lot-103011 Temperature 29.0 1:10 Zeta 3(° C.): Dispersant Name: Water Count Rate (kcps): 163.5 Viscosity (cP):0.8096 Zeta Runs: 12 Dispersant RI: 1.330 Attenuator: 6 MonomodalAnalysis Results Result Quality: Good Mobility −2.383 (μmcm/Vs): Zeta−28.2 Standard Deviation 1.876 Potential (mV): (μmcm/Vs): Standard 22.2Conductivity 2.48 Deviation (mV): (mS/cm): QualityFactor: 3.15Multimodal Distribution Mean (mV) Area (%) Width (mV) Peak 1: −26.1100.0 6.52 Peak 2: 0.00 0.0 0.00 Peak 3: 0.00 0.0 0.00

Parameters for FIGS. 3A and 3B

Measurement Details Sample Name: Lot-103011 Temperature 29.0 1:10 Zeta 4(° C.): Dispersant Name: Water Count Rate (kcps): 164 Viscosity (cP):0.8096 Zeta Runs: 12 Dispersant RI: 1.330 Attenuator: 6 MonomodalAnalysis Results Result Quality: — Mobility −1.891 (μmcm/Vs): Zeta −22.4Standard Deviation 2.354 Potential (mV): (μmcm/Vs): Standard 27.9Conductivity 2.54 Deviation (mV): (mS/cm): QualityFactor: 1.45Multimodal Distribution Mean (mV) Area (%) Width (mV) Peak 1: −25.8 49.65.73 Peak 2: −11.1 0.00 6.77 Peak 3: −47.3 3.4 3.83

Parameters for FIGS. 4A and 4B

Measurement Details Sample Name: Lot-043012-A TALP Temperature 29.0buffer 1:10 Z . . . (° C.): Dispersant Name: Water Count Rate (kcps):183.1 Viscosity (cP): 0.8096 Zeta Runs: 12 Dispersant RI: 1.330Attenuator: 7 Monomodal Analysis Results Result Quality: Good Mobility−2.074 (μmcm/Vs): Zeta −24.6 Standard Deviation 0.6904 Potential (mV):(μmcm/Vs): Standard 8.18 Conductivity 1.80 Deviation (mV): (mS/cm):QualityFactor: 3.54 Multimodal Distribution Mean (mV) Area (%) Width(mV) Peak 1: −24.6 100 8.18 Peak 2: 0.00 0.0 0.00 Peak 3: 0.00 0.0 0.00

Parameters for FIGS. 5A and 5B

Measurement Details Sample Name: Lot-043012-A MRS Temperature 29.0buffer 1:10 Z . . . (° C.): Dispersant Name: Water Count Rate (kcps):146.5 Viscosity (cP): 0.8096 Zeta Runs: 12 Dispersant RI: 1.330Attenuator: 6 Monomodal Analysis Results Result Quality: Good Mobility−1.392 (μmcm/Vs): Zeta −16.5 Standard Deviation 0.5526 Potential (mV):(μmcm/Vs): Standard 6.55 Conductivity 2.28 Deviation (mV): (mS/cm):QualityFactor: 3.74 Multimodal Distribution Mean (mV) Area (%) Width(mV) Peak 1: −16.5 100 6.55 Peak 2: 0.00 0.0 0.00 Peak 3: 0.00 0.0 0.00

Parameters for FIGS. 6A and 6B

Measurement Details Sample Name: Lot-043012-A dH2O Temperature 29.0 1:10Zeta 2 (° C.): Dispersant Name: Water Count Rate (kcps): 70.4 Viscosity(cP): 0.8096 Zeta Runs: 12 Dispersant RI: 1.330 Attenuator: 6 MonomodalAnalysis Results Result Quality: Good Mobility −2.244 (μmcm/Vs): Zeta−26.6 Standard Deviation 6.039 Potential (mV): (μmcm/Vs): Standard 71.6Conductivity 0.0331 Deviation (mV): (mS/cm): QualityFactor: 2.31Multimodal Distribution Mean (mV) Area (%) Width (mV) Peak 1: −0.594 1006.89 Peak 2: 0.00 0.0 0.00 Peak 3: 0.00 0.0 0.00

Parameters for FIGS. 7A and 7F

Sample Details Sample Name: 041212-B-Tris Zeta Mean SOP Name: Zetastandard 3 × 20 25 C. no wait.sop File Name: 2012-01020 new DispersantWater measure . . . Name: Record Number: 1659 Dispersant RI: 1.330Measurement Friday, Viscosity (cP): 0.8872 Date and Apr. 20, 2012 Time:2:55:0 . . . System Temperature 25.0 Zeta Runs: 20 (° C.): Count Rate2456.7 Measurement 2.00 (kcps): Position (mm): Cell Clear disposableAttenuator: 9 Description: zeta cell Results Result Quality: Good ZetaPotential (mV): −42.2 Zeta SD (mV): 5.70 Mobility (μmcm/Vs): −3.307Mobility SD (μmcm/Vs): 0.4464 Wall Zeta Potential (mV): −47.9 EffectiveVoltage (V): 151 Conductivity (mS/cm): 0.240 Mean (mV) Area (%) Width(mV) Peak 1: −42.2 100 5.64 Peak 2: 0.00 0.0 0.00 Peak 3: 0.00 0.0 0.00

To the extent that the term “includes” or “including” is used in thespecification or the claims, it is intended to be inclusive in a mannersimilar to the term “comprising” as that term is interpreted whenemployed as a transitional word in a claim. Furthermore, to the extentthat the term “or” is employed (e.g., A or B) it is intended to mean “Aor B or both.” When the applicants intend to indicate “only A or B butnot both” then the term “only A or B but not both” will be employed.Thus, use of the term “or” herein is the inclusive, and not theexclusive use. See Bryan A. Garner, A Dictionary of Modern Legal Usage624 (2d. Ed. 1995). Also, to the extent that the terms “in” or “into”are used in the specification or the claims, it is intended toadditionally mean “on” or “onto.” To the extent that the term“selectively” is used in the specification or the claims, it is intendedto refer to a condition of a component wherein a user of the apparatusmay activate or deactivate the feature or function of the component asis necessary or desired in use of the apparatus. To the extent that theterms “coupled” or “operatively connected” are used in the specificationor the claims, it is intended to mean that the identified components areconnected in a way to perform a designated function. To the extent thatthe term “substantially” is used in the specification or the claims, itis intended to mean that the identified components have the relation orqualities indicated with degree of error as would be acceptable in thesubject industry.

As used in the specification and the claims, the singular forms “a,”“an,” and “the” include the plural unless the singular is expresslyspecified. For example, reference to “a compound” may include a mixtureof two or more compounds, as well as a single compound.

As used herein, the term “about” in conjunction with a number isintended to include ±10% of the number. In other words, “about 10” maymean from 9 to 11.

As used herein, the terms “optional” and “optionally” mean that thesubsequently described circumstance may or may not occur, so that thedescription includes instances where the circumstance occurs andinstances where it does not.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group. As will beunderstood by one skilled in the art, for any and all purposes, such asin terms of providing a written description, all ranges disclosed hereinalso encompass any and all possible sub-ranges and combinations ofsub-ranges thereof. Any listed range may be easily recognized assufficiently describing and enabling the same range being broken downinto at least equal halves, thirds, quarters, fifths, tenths, and thelike. As a non-limiting example, each range discussed herein may bereadily broken down into a lower third, middle third and upper third,and the like. As will also be understood by one skilled in the art alllanguage such as “up to,” “at least,” “greater than,” “less than,”include the number recited and refer to ranges which may be subsequentlybroken down into sub-ranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember. For example, a group having 1-3 cells refers to groups having 1,2, or 3 cells. Similarly, a group having 1-5 cells refers to groupshaving 1, 2, 3, 4, or 5 cells, and so forth. While various aspects andembodiments have been disclosed herein, other aspects and embodimentswill be apparent to those skilled in the art.

As stated above, while the present application has been illustrated bythe description of embodiments thereof, and while the embodiments havebeen described in considerable detail, it is not the intention of theapplicants to restrict or in any way limit the scope of the appendedclaims to such detail. Additional advantages and modifications willreadily appear to those skilled in the art, having the benefit of thepresent application. Therefore, the application, in its broader aspects,is not limited to the specific details, illustrative examples shown, orany apparatus referred to. Departures may be made from such details,examples, and apparatuses without departing from the spirit or scope ofthe general inventive concept.

The various aspects and embodiments disclosed herein are for purposes ofillustration and are not intended to be limiting, with the true scopeand spirit being indicated by the following claims.

1. A method for magnetic cellular manipulation, the method comprising:contacting sperm cells with a plurality of magnetic particles, eachmagnetic particle in the plurality of magnetic particles being nogreater than 1000 nm in size and each magnetic particle comprising anegative zeta potential charge and a chargeable silicon-containingcompound, the magnetic particles binding to the sperm cells through anelectrical charge interaction; and manipulating the bound magneticparticles with a magnetic field.
 2. The method of claim 1, each magneticparticle comprising Fe₃O₄.
 3. The method of claim 1, the chargeablesilicon-containing compound comprising2-(carbomethoxy)ethyltrimethoxysilane.
 4. The method of claim 1, furthercomprising the step of diluting the sperm cells with a buffered media.5. The method of claim 1, each magnetic particle being between 30 nm to1000 nm in size.